Regulation of species metabolism in synthetic community systems by environmental pH oscillations

Constructing a synthetic community system helps scientist understand the complex interactions among species in a community and its environment. Herein, a two-species community is constructed with species A (artificial cells encapsulating pH-responsive molecules and sucrose) and species B (Saccharomyces cerevisiae), which causes the environment to exhibit pH oscillation behaviour due to the generation and dissipation of CO2. In addition, a three-species community is constructed with species A′ (artificial cells containing sucrose and G6P), species B, and species C (artificial cells containing NAD+ and G6PDH). The solution pH oscillation regulates the periodical release of G6P from species A′; G6P then enters species C to promote the metabolic reaction that converts NAD+ to NADH. The location of species A′ and B determines the metabolism behaviour in species C in the spatially coded three-species communities with CA′B, CBA′, and A′CB patterns. The proposed synthetic community system provides a foundation to construct a more complicated microecosystem.

there are major issues with this manuscript that need to be addressed.First of all, I don't understand the oscillation between pH 6.5 and 6.4.The viscosity change and subsequent difference in release of sucrose is very small in this window, and since the reset (release of CO2 from the system) is the slowest process, there is no reason why not a steady state should be obtained in which, due to differences in rates, the system finds a pH in which production and release are in balance.Obtaining oscillations is notoriously difficult as the different rates have to be matched very well.It is highly surprising that under all measured conditions oscillations take place.Why do you still see oscillations at pH 7, when there is no gelation effect?The authors should therefore study the individual rates of the different steps in more detail in order to better understand how this system really works.Furthermore, why does the pH increase also take longer in subsequent cycles?This is a pure physical process (release from CO2 to the air) and should not be affected by the concentration of sucrose.Secondly, the pH of a CO2 saturated system should be more close to 4 at atmospheric pressure.It is therefore not logical that the pH increases that effectively.The authors did not perform a control reaction in which they use only yeast cells and vary the sucrose concentration in the window that is achievable by the release from artificial cell A. I find it difficult to see how the closely packed cell populations could prevent diffusion of small molecules or protons.The authors should study this in more detail with e.g.model dye components.
Reviewer #3: Remarks to the Author: Han et al. tried to construct unique artificial cells and create cell communities consisting of artificial cells and natural cells.The core technique to accomplish this research is the use of pH-sensitive molecule that phase-shift between fluid and gel phases, and encapsulate it inside giant vesicles.By changing the surrounding pH, the artificial cells release sugar into the environment and the neighbor yeasts consume the sugar and release CO2 into the environment.As a consequence, the oscillation of pH change was generated in the society.The authors developed the society more complicated by increasing the species as 2 types of artificial cells and yeasts.Finally, the authors demonstrate the controlled communication between the species by giving spatial constraints.The concept of an artificial community with artificial cells and natural cells is previously proposed by Mansy et al. (2014) and Stano et al. (2018).But this work is further advanced with fine-tuning.This would be a noteworthy paper in the field of artificial cell research which is rapidly developing nowadays.The data are clear and analyzed well.Figures are well arranged to easily understand the results.But, unfortunately, the description in the Discussion is poor.The authors just repeat the explanation of what they did but mentioned nothing further.The authors should describe, at least, how the artificial system achieved in this work will benefit the study of biological phenomena, or how does it useful for controlling the biological system.Therefore, before accepting the paper, the reviewer believes that the discussion part should be enriched so that the readers can understand what value this work has for life science as a whole.

Response to the reviewers' comments
For the sake of clarity, the comments of the reviewer have been collated in black, and our response to each comment appears in blue.All the changes to the manuscript are highlighted in red.

Li et al produce what they refer to as artificial cells that can interact with yeast and another type of artificial cell. Importantly, feedback cycles were possible between the artificial cells and yeast. The artificial cell was simply a phospholipid vesicle containing "pH responsive molecules" and sucrose. The membrane possessed melittin pores. At low pH, a gel formed, whereas at higher pH, the solution was less viscous. Released sucrose (at high pH) was consumed by the yeast, leading to a decrease in pH through the release of CO2 (carbonate). The low pH blocked the release of more sucrose.
Over time, through equilibration with the atmosphere, the pH increased again, leading to more sucrose being released and the commencement of another cycle.

1) The cyclic nature of the interaction of the artificial cells and the yeast was to me the advance in the work. However, I don't consider what was built an artificial cell. It's much too simplistic. There's not even nucleic acid involved. Nothing is genetically encoded.
Thank the reviewer for the comment.The artificial cells refer to the structures mimicking partial/whole cell structure and functions.The cell structures include membrane, organelles, cytosol etc.The cell functions are metabolism, division, mass transport across the membrane, etc.The phospholipid vesicles containing non-biological components or enzymes were called artificial cells ( Nat.Commun.2014, 5, 5305; PNAS 2019, 116, 16711; Nat.Chem.Biol.2018, 14, 86) in this field.We call our phospholipid vesicle structures as artificial cells because they mimicked the cell function of mass transport across the membrane, as well as the cell responsive function to environmental stimulus.We added the description of artificial cells in the introduction as below in page 3.

2) It is nice that a pH responsive system was built, but that's not very novel. There are many publications on pH responsive hydrogels and coacervates.
Thank the reviewer for the comment.Most of the current pH responsive systems were regulated by the environment, which exhibited one-way response manner.To the best of our knowledge, it is the first time to encapsulate pH responsive hydrogel in phospholipid vesicles, which communicate the environment in a bidirectional response manner via the interaction with yeasts, consequently to generate pH oscillation of solution.

3) It also was not clear from the main text what molecules were used. I don't think the pH responsive molecule was ever named, although the structure was given in Fig. S1.
Thank the reviewer for the comment.We have named the molecules and modified the text as below in page 21.

4) Similarly, the properties of melittin were never explained.
Thank the reviewer for the comment.We have added a description of the properties of melittin in the main text as below in page 4.
[…] The pH-responsive molecules changed from fluid phase at high pH value (≥7) into gel phase at low pH value (≤6) due to the hydrogen bonds (Fig. 1a and b).Melittin is a 26-amino-acid αhelical peptide, which forms pores on the lipid bilayer membrane for substance exchange between internal and external of lipid vesicles. 35,36Here, the melittin nanopores on the bilayer membrane allowed protons exchange between inner artificial cells and external environments.[…]

5) The need for gadobutrol was not explained (nor what gadobutrol was).
Thank the reviewer for the comment.We have added a description of gadobutrol and its necessity as below in page 15.
[…] Spatial coded three-species-communities were constructed inside each well of a steel mesh (Supplementary Fig .16) under a magnetic field using Magneto-Archimedes principle with a homemade device 37,38 (Supplementary Fig .17).Magneto-Archimedes effect was determined by equation (2) 39 : where U (r) is the static magnetic potential energy of GUVs with radius R at r position, μ0 is the magnetic permeability of vacuum, χG and χS are the magnetic susceptibility of GUV and solution respectively, H (r) is the magnetic field at r position.If χG is less than χS, the GUV in the solution tends to move towards the region with the weakest magnetic field to lower U (r). Gadobutrol is a paramagnetic contrast agent used in magnetic resonance imaging. 40The addition of gadobutrol aimed to increase χS.Three patterns (CA′B, CBA′, A′CB) of three-species-communities were obtained by varying addition order of species A′, species B, and species C (Supplementary Fig .18). […] 6) Some of the information should probably be moved to the supplemental section, including Fig. 2b and Fig. 3.
Thank the reviewer for the comment.We have moved original Fig. 2b and Fig. 3 to the supplemental section as Supplementary Fig .5, Supplementary Fig .9 and Supplementary Fig .12, respectively.

7) The three species system consists of an extra "artificial cell" that receives released glucose 6phosphate and then with an enzyme (which wasn't explained) reduced NAD+ to NADH, but this extra artificial cell did not seem to add anything to the story. What did we learn from this?
Thank the reviewer for the comment.We supplemented the descriptions of NADH and G6PDH in the main text.Glucose-6-phosphate dehydrogenase (G6PDH) is an important enzyme involved in glycolysis pathway.G6PDH inside species C can convert glucose-6-phosphate and NAD + to 6-phosphate gluconate lactone and NADH respectively.Therefore, species C is an artificial cell for metabolic mimicry.The introduce of species C increased the complexity of communities from twospecies-community to three-species-community.The biochemical reaction in species C was adjusted by the communications between species A′ and B. The species A′, B, C and environment were an integral system, among which these elements activities were influenced by others.We established a three-species-community with complex internal dynamic interactions, which helps to understand the behavior of complex community.We modified the main text as below in pages 12 and 13.
[…] The controlled release of G6P from species A′ was expected to trigger the stepped generation of NADH inside species C (Fig. 3a).Glucose-6-phosphate dehydrogenase (G6PDH) is an important enzyme involved in glycolysis pathway.Upon receiving G6P, the G6PDH inside species C converted G6P and NAD + to 6-phosphate gluconate lactone and NADH, respectively.NADH is important biomoleculeinvolved in many metabolic pathways.Species C is an artificial cell capable of metabolism mimicry.
[…] No NADH was generated inside species C with the absence of G6PDH in species C (Fig. 3e, blue curve), NAD + in species C (Fig. 3e, black curve) or G6P in species A (Fig. 3e, red curve), which confirmed the feedback between species A′ and species B regulated the internal ′metabolism′ in species C. The species A′, B, C and environment were an integral system, among which these elements activities were influenced by others.We established a three-species-community with complex internal dynamic interactions, which helps to understand the behavior of complex community.

8) The spatial control of the location of the participating artificial cells is certainly "cool" but the data did not seem to tell us anything more than we would have already guessed.
Thank the reviewer for the comment.The spatial distribution of cells plays a crucial role for cell function to the community.Here, we have demonstrated for the first time the importance of spatial location distribution in synthetic communities using a three-species-community constructed using magnetic fields.We found the spatial distribution of species in a community influenced their internal interactions, which helps to understand the structure of communities.In addition, we performed dye diffusion experiments in different spatial coded communities using fluorescein as a model dye to observe the inhibitory effect of tightly packed species on the diffusion of small molecules more intuitively.We modified the text as below in pages 16,18 and 24.
[…] The intensity of community A′CB (Fig. 4i) continuous increased, because the release of G6P from species A′ at relative high pH (Fig. 4g) diffused into the adjacent species C to generate NADH.In order to observe the inhibitory effect of tightly packed species on the diffusion of small molecules more intuitively, we prepared a species A′′ encapsulating model dye molecule of fluorescein (Mw=332.3g/mol), which has green fluorescence and molecular weight similar to sucrose (Mw=342.3g/mol) (Supplementary Fig .21a).Two three-species communities were constructed by arranging species A′′, species B (Supplementary Fig .21b), and species C (labeled with TR DHPE, Supplementary Fig .21c) in the order of A′′CB (Supplementary Fig .21d, e, and f) and CBA′′ Supplementary Fig .21h, i, and j).No green fluorescence was observed in the species B region in A′′CB community within 270 minutes (Supplementary Fig .21g) due to the close pack of species C. Similarly, no green fluorescence was observed in the species C region due to the close pack of species B (Supplementary Fig .21k).All abovementioned results demonstrated that the spatial distribution of species in the community did exhibit dramatic impact on the behavior of community system.
[…] The location of species in the spatial coded three-species-community system is confirmed to have dramatic impact on the interaction among species and between community and its environment.
The species in natural communities often exist in spatial orders.The influence study of spatial distribution on signal transmission among species in spatial coded synthetic communities helps to understand the structure and function of communities.
[…] By varying the addition order of pH-responsive artificial cells containing sucrose and G6P (species A′), yeasts (species B) and artificial cells containing NAD + and G6PDH (species C), A′CB, CA′B and CBA′ were obtained.For dye penetration experiments, species A′′ (containing G6P, fluorescein and sucrose), species B and species C were coded to obtain A′′CB and CBA′′.To prevent GUVs from breaking during the experiment, 300 μL of DPPC ethanol-water solution (0.10 mg/mL, ethanol: water = 40 %:60 %, v/v) was added to coverslips, followed by heating at 60 °C for 10 min and washing 3 times with 400 mM gadobutrol solution.

9) For me, the "two species" system is interesting and seems like a nice platform from which more could be built, but I also do not think that this is a big advancement from previous work.
Thank the reviewer for the comment.The advancement of our work is the involvement of environment (solution) in the community system, rather than only taking into accounts of species.More importantly, the solution pH oscillation (environment variation) caused by two species influenced the ′metabolic pathway′ of the third species.Therefore, we established a community possessing complex dynamic interaction network, which provide a way for investigating complicated inter network reaction among micro-ecosystems, and laid the foundation for building more complex systems in the future to perform higher-order functions.We modified the main text as below in pages 12 and 18.
[…] the balance of CO2 dissipation rate and CO2 production rate caused lower steady median pH value, bigger amplitude and shorter period of oscillation at higher percentage of species A.
The construction of a two-species-community oscillation system provides the possibility of constructing complex dynamic response systems using simple components.Although two-speciescommunity is a useful platform for studying inter species interactions, natural communities typically consist of more than two species to maintain community stability.In the following content, a threespecies community was constructed to investigated the interactions among species and environment.
[…] The location of species in the spatial coded three-species-community system is confirmed to have dramatic impact on the interaction among species and between community and its environment.
The species in natural communities often exist in spatial orders.The influence study of spatial distribution on signal transmission among species in spatial coded synthetic communities helps to understand the structure and function of communities.

10) I would strongly suggest to the authors during revisions to more clearly and thoroughly explain how the experiments were run, the purpose of the different molecules that were used, and what can be learned from three species and spatial control experiments.
Thank the reviewer for the comment.We have added more experimental details, experimental design, and the significance of three species and spatial control experiments as below in pages 4, 12, 13, 15 and 18.
Page 4 […] The pH-responsive molecules changed from fluid phase at high pH value (≥7) into gel phase at low pH value (≤6) due to the hydrogen bonds (Fig. 1a and b).Melittin is a 26-amino-acid αhelical peptide, which forms pores on the lipid bilayer membrane for substance exchange between internal and external of lipid vesicles. 35,36Here, the melittin nanopores on the bilayer membrane allowed protons exchange between inner artificial cells and external environments. […] Page 12 […] The controlled release of G6P from species A′ was expected to trigger the stepped generation of NADH inside species C (Fig. 3a).Glucose-6-phosphate dehydrogenase (G6PDH) is an important enzyme involved in glycolysis pathway.Upon receiving G6P, the G6PDH inside species C converted G6P and NAD + to 6-phosphate gluconate lactone and NADH, respectively.NADH is important biomolecule to provide reductive force in many metabolic pathways.Species C is an artificial cell capable of metabolism mimicry.
Page 13 […] No NADH was generated inside species C with the absence of G6PDH in species C (Fig. 3e, blue curve), NAD + in species C (Fig. 3e, black curve) or G6P in species A (Fig. 3e, red curve), which confirmed the feedback between species A′ and species B regulated the internal ′metabolism′ in species C. The species A′, B, C and environment were an integral system, among which these elements activities were influenced by others.We established a three-species-community with complex internal dynamic interactions, which helps to understand the behavior of complex community.
Page 15 […] Spatial coded three-species-communities were constructed inside each well of a steel mesh (Supplementary Fig .16) under a magnetic field using Magneto-Archimedes principle with a homemade device 37,38 (Supplementary Fig .17).Magneto-Archimedes effect was determined by equation ( 2) 39 : where U (r) is the static magnetic potential energy of GUVs with radius R at r position, μ0 is the magnetic permeability of vacuum, χG and χS are the magnetic susceptibility of GUV and solution respectively, H (r) is the magnetic field at r position.If χG is less than χS, the GUV in the solution tends to move towards the region with the weakest magnetic field to lower U (r). Gadobutrol is a paramagnetic contrast agent used in magnetic resonance imaging. 40The addition of gadobutrol aimed to increase χS.Three patterns (CA′B, CBA′, A′CB) of three-species-communities were obtained by varying addition order of species A′, species B, and species C (Supplementary Fig .18). […] Page 18 […] The location of species in the spatial coded three-species-community system is confirmed to have dramatic impact on the interaction among species and between community and its environment.
The species in natural communities often exist in spatial orders.The influence study of spatial distribution on signal transmission among species in spatial coded synthetic communities helps to understand the structure and function of communities.

Reviewer #2 (Remarks to the Author): This report describes the use of artificial cells and yeast cells to develop an oscillating system based on a feedback mechanism. The pH of the medium oscillates around 6.4, which is thought to be the result of the gelation-dependent release of sucrose and the subsequent production of CO2.
A third population is added to the system, in which the positioning of the three populations has an effect on the overall activity.The construction of artificial cell populations with interactive mechanisms is of interest to the synthetic cell community.The combination with yeast cells is not often observed.Still there are major issues with this manuscript that need to be addressed.

1) First of all, I don't understand the oscillation between pH 6.5 and 6.4. The viscosity change and subsequent difference in release of sucrose is very small in this window, and since the reset (release of CO2 from the system) is the slowest process, there is no reason why not a steady state should be obtained in which, due to differences in rates, the system finds a pH in which production and release are in balance. Obtaining oscillations is notoriously difficult as the different rates have to be matched very well. It is highly surprising that under all measured conditions oscillations take place. Why do you still see oscillations at pH 7, when there is no gelation effect? The authors should therefore study the individual rates of the different steps in more detail in order to better understand how this system really works.
Thank the reviewer for the comment.To better understand how this oscillating system works, we studied the effect of pH on the leakage of sucrose from artificial cells by measuring the concentrations of sucrose released to the external solution at different pH (Fig. R1a).A new parameter ′relative diffusion coefficient′ was extracted to describe the leaking ability of sucrose driven by the concentration difference of the inner and outer membrane, showing that there was a gelation effect at a wide range of pH from 6 to 7 (Fig. R1b).Although the diffusion difference was very small, the coupled nonlinear processes may amplify this difference, leading to continuous oscillations.The oscillation range of T1 period in Fig. 2b is 6.63 to 6.42, which is closer to 6.6 and 6.4.The pHoscillating system was the result of coupled multiple nonlinear processes, which constructed a closed feedback loop, where the negative feedback motif from H + to sucrose (Fig. R2a) played an important role in oscillation generation.Fig. R2a is the scheme of non-linear feedback network in the oscillation system.In order to further investigate the contribution of the diffusion difference to the oscillating system, we simplified the complex system to a simple oscillation model based on the binary function (see below ′Oscillation computational model I′ section), where the relative diffusion coefficient of sucrose was set 3×10 -13 cm 2 /s in the case of pH greater than 6.6, while it was set 0 in the case of pH less than 6.6.The setting value of 3×10 -13 cm 2 /s was the diffusion difference between pH 6.6 and 6.4.We found that oscillation can be generated under this condition (Fig. R2b, black curve), but when its difference was reduced to 0.8×10 -13 cm 2 /s, the system would reach a steady state (Fig. R2b, red curve), indicating that the pH-responsive diffusion differences could be amplified to generate pH-responsive oscillations through multiple nonlinear processes.
In addition, the appearance of oscillations was the clever coupling of different parameters during the process, where the escape of CO2 was also a critical part.Open system was another necessary condition for continuous oscillations.But it is hard to calculate the certain value of mass transfer coefficient of CO2, because there were multiple complicate influencing factors in this process.However, from the simulation, we guess that the mass transfer coefficient of CO2 can be coupled with other parameters in a relatively wide range (0.01 cm/s ~ 1×10 -10 cm/s) to obtain oscillations (Fig. R2c).
Fig. R2.a, The scheme of non-linear feedback network in the oscillation system.b, Time-dependent plot of pH under relative diffusion coefficient of 3×10 -13 cm 2 /s (black curve) and 0.8×10 -13 cm 2 /s (red curve) obtained by oscillation computational model I.The relative diffusion coefficient of sucrose was set as 3×10 -13 cm 2 /s and 0.8×10 -13 cm 2 /s in the case of pH greater than 6.6, while it was set as 0 in the case of pH less than 6.6.was set as 0.01 cm/s.c, Time-dependent plot of pH under different mass transfer coefficient in oscillation computational model I.
was set to be 0.01 and 1×10 -10 cm/s, respectively.The relative diffusion coefficient of sucrose was set as 1×10 -12 cm 2 /s.
The reason for oscillation at pH 7 is that gelation effect still exists around pH 7. The viscosities of internal artificial cell are 5.09×10 -3 mPa•s at pH 7 and 1.47×10 -3 mPa•s at pH 8. (Supplementary Fig .2) Considering the process of sucrose releasing from the artificial cells was influenced by multiple factors, such as the membrane permeability besides viscosity, we introduce a parameter ′relative diffusion coefficient′ of sucrose to qualitatively study the effect of pH on sucrose leakage (Fig. R1b).A diffusion difference of sucrose leakage around pH 7 was observed (Fig. R1b).Through the oscillation model I, in which the binary function was performed as relative diffusion coefficient of sucrose was set as 1.5×10 -13 cm 2 /s in the case of pH greater than 7, while it was set as 0 in the case of pH less than 7.The simulation confirmed the oscillation could be generated under this condition (Fig. R3).

The introduction of relative diffusion coefficient
The new parameter ′relative diffusion coefficient′ was introduced to describe the rate of sucrose releasing out of pH-responsive artificial cells.We simplified the process of molecule diffusion to one driven by the concentration difference of the inner and outer membrane.The relative diffusion coefficient was determined by Fick's equation: The finite explicit approach was used to simplify equation ( 1): where [sucrose]in was the concentration of sucrose inside the artificial cells; [sucrose]out was the concentration of sucrose outside the artificial cells; Dsucrose′ denoted relative diffusion coefficient of sucrose; ΔT was the time interval, and d represented the diffusion distance determined by the concentration of artificial cells.Here d was set as 6 μm with artificial cells concentration of 3.7×10 8 cells/mL and cells size of 5 μm.
The Boltzmann model was used to fit the relationship between the relative diffusion coefficient of sucrose and the environment solution pH: Oscillation computational model I In the system, sucrose-entrapped artificial cells and yeasts were assumed to be dispersed homogenously.At high environment solution pH, the membrane of artificial cells showed high permeability to sucrose, which were consumed by yeasts to generate CO2, leading to the decrease of environment solution pH.In this case, sucrose stopped releasing.As CO2 escaped into the air, the environment solution pH has rebounded apparently, triggering next round of pH oscillations.
The leakage of sucrose from artificial cells was simplified to be a binary function, where the relative diffusion coefficient of sucrose was set as 3×10 -13 cm 2 /s in the case of pH greater than 6.6, while it was set as 0 in the case of pH less than 6.6.The concentration of sucrose, CO2, and H + in the solution were assumed to be uniform, because their diffusion (D (sucrose) = 1.86×10 -6 cm 2 /s ( J. Phys.Chem.A, 2003, 107 (6), 936-943); D (CO2) =1.85×10 -5 cm 2 /s(J.Agric.Food Chem., 2003, 51 (26), 7560-7563) were too fast, compared to the process of sucrose leaking out of artificial cells.
The escape of CO2 into the air can be described based on two-film theory: Here, N was represented as mass transfer flux; K was represented as mass transfer coefficient, and set as 0.01, which was just a hypothetical value; [CO2]in was the concentration of CO2 in the solution; [CO2]out was the concentration of CO2 in the air.
2) Furthermore, why does the pH increase also take longer in subsequent cycles?This is a pure physical process (release from CO2 to the air) and should not be affected by the concentration of sucrose.
Thank the reviewer for the comment.The extension of the oscillation period was attributed to the reduction of the sucrose concentration difference between the inner and outer membrane.Due to the continuous release of sucrose, the concentration difference decreased in the subsequent cycles, resulting in the slower production of H + .In the pH-rising phase of oscillation cycle, CO2 was continuously produced in the system.Therefore, it wasn't a pure physical process of CO2 releasing to the air.
To support this view, we constructed a simple model that abstracted the oscillating system into a nonlinear feedback network, where the introduction of k1′ simulated the self-producing ability of sucrose, which came from the diffusion ability of sucrose from artificial cells.(Fig. R4a) The rise of internal concentration of sucrose or density of artificial cells would be performed as the increase of k1′.By setting larger value of k1′ in the simulation, we found that the amplitude would increase while the period would decrease, which echoed the experimental results, proving the feasibility of the simple model (Fig. R4b).In the improvement of the model, we set k1′ as a parameter that gradually decreased over time, due to the continuous release of sucrose from the artificial cells in the subsequent cycles.The oscillation periods were observed gradually longer (Fig. R4c), confirming that the extension of the oscillation cycle was the result of the reduction of the sucrose concentration difference.Meanwhile the pH increase took longer in the subsequent cycles.

Oscillation computational model II
In order to investigate the influencing factors of oscillation period, we developed another oscillation computational model based on the negative feedback loop.The whole reactions could be described as below: where k1′ was set as 1; k2′ was set as 1; k3′ was set as 1; k4′ was set as 0.01; k5′ was set as 0.1.c, Time-dependent plot of the negative number of the logarithm of ′C′ concentrations in oscillation computational model II, where k1′ = 2 -0.02t; k2′ = 1; k3′ = 0.7; k4′ = 0.01; and k5′ = 0.1.
We modified the main text as below in pages 8.
[…] It can be explained by the consumption of sucrose inside species A, which resulted the difference of the concentration of solution components at each equilibrium point in subsequent cycles.The decrease of the sucrose concentration difference between the inner and outer membrane slowed down the production of H + and the release of CO2, thereby exhibiting longer oscillation period in the subsequent cycles.The longest oscillation can exist for 960 min due to the completely consumed of sucrose in species A (Fig. 2d, Supplementary Fig .6). […] 3) Secondly, the pH of a CO2 saturated system should be more close to 4 at atmospheric pressure.It is therefore not logical that the pH increases that effectively.
Thank the reviewer for the comment.We measured the pH of pure water in the air for 60 h, indicating that the pH of air CO2 saturated water solution is 5.8.However, with continuous purging CO2 into the solution, the solution pH becomes 3.99, which is lower than that of air CO2 saturated water solution.In terms of our solution (400 mM gadobutrol), the air CO2 saturated pH value is 7.25 after continuous monitoring for 60 h.With purging CO2 into our solution, the pH value is 5.21.
Because the pH value of air CO2 saturated gadobutrol solution is 7.25, the CO2 produced by yeasts dissipate into the air, leading to an increase of solution pH.

4) The authors did not perform a control reaction in which they use only yeast cells and vary the sucrose concentration in the window that is achievable by the release from artificial cell A.
Thank the reviewer for the comment.We carried out the suggested experiments, and added below contents in pages 11 and 23.
[…] The faster CO2 dissipation rate balanced CO2 production rate, which explained the lower steady median pH value, the bigger amplitude and shorter average period at higher sucrose concentration in species A. In order to confirm the necessity of pH responsive release of sucrose from species A to the oscillation phenomenon, the solution pH variation of direct mixing sucrose with yeasts were measured (Supplementary Fig .10).The sucrose concentrations leaking out from species A (containing 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM sucrose) for 30 minutes (Supplementary Fig .10a) were chosen to mix with yeasts.No solution pH oscillations were observed (Supplementary Fig .10b).The solution pH dropped in the first phase, and gradually increased in the second phase.

Therefore, before accepting the paper, the reviewer believes that the discussion part should be enriched so that the readers can understand what value this work has for life science as a whole.
Thank the reviewer for the comment.We added the descriptions to emphasize the significance of our system as below.The mentioned papers were cited in the manuscript.
Page 12 […] The controlled release of G6P from species A′ was expected to trigger the stepped generation of NADH inside species C (Fig. 3a).Glucose-6-phosphate dehydrogenase (G6PDH) is an important enzyme involved in glycolysis pathway.Upon receiving G6P, the G6PDH inside species C converted G6P and NAD + to 6-phosphate gluconate lactone and NADH, respectively.NADH is important biomolecule involved in many metabolic pathways.Species C is an artificial cell capable of metabolism mimicry.
Page 13 […] No NADH was generated inside species C with the absence of G6PDH in species C (Fig. 3e, blue curve), NAD + in species C (Fig. 3e, black curve) or G6P in species A (Fig. 3e, red curve), which confirmed the feedback between species A′ and species B regulated the internal ′metabolism′ in species C. The species A′, B, C and environment were an integral system, among which these elements activities were influenced by others.We established a three-species-community with complex internal dynamic interactions, which helps to understand the behavior of complex community.

Page 18
The construction of synthetic communities plays an important role in studying the interaction between species and the environment or understanding the complex behavior among populations.Three artificial cells are built by encapsulating sucrose/pH-responsive molecule (species A), sucrose/pH-responsive molecule/G6P (species A′), and NAD + (species C), respectively.Yeasts are species B. A two-species community is constructed using species A and species B, which causes pH oscillation of its environment by the balance of sucrose consumption by yeasts and CO2 dissipation from solution.The sucrose released from species A is consumed by yeast (species B) to generates CO2 to decrease solution pH, whilst the oversaturated CO2 dissipates into the air to increase solution pH.The pH oscillation behavior is influenced by the initial sucrose concentration in species A and the ratio between species A and B. The higher initial sucrose concentration causes lower steady median pH values, bigger amplitude and shorter period.The higher ratio of species A to B results in lower steady median pH values, bigger amplitude and shorter period.Significantly, the pH oscillation between species A′ and B in a three-species-community system regulates the periodical release of G6P from species A′, which tunes the 'metabolic' reaction from NAD + to NADH inside species C. The location of species in the spatial coded three-species-community system is confirmed to have dramatic impact on the interaction among species and between community and its environment.The species in natural communities often exist in spatial orders.The influence study of spatial distribution on signal transmission among species in spatial coded synthetic communities helps to understand the structure and function of communities.
The signal communication among species in nature communities has dynamic characteristics.By responding to environmental changes or signal molecules secreted by other species, species in the community can regulate their metabolism to better adapt to the environment and improve their survival ability.Two types of artificial cells were developed with the functions of environmental stimulus response (species A) and metabolism mimicry (species C).The environment as a key element was dynamically involved in the system to influence the function of species in constructed communities.The complex interactions among species and environment were demonstrated in the two-species/three-species communities.Therefore, we established communities possessing complex dynamic interaction network, which provide a way for investigating complicated inter network reaction among micro-ecosystems, and laid the foundation for building more complex systems in the future to perform higher-order functions.Thank the reviewer for the comment.We modified the head title of Fig. 2 as below.Live yeasts (species B) were stained with FDA (green, in Q3), and dead yeasts were stained with PI (red, in Q1).Total number of particles counted was10,000.
[…] The CO2 dissipation rate was faster at lower pH, since the slops from the first lowest point to the first highest point were bigger at higher sucrose concentration (Fig. 3b, purple dashed boxes) and the tangents of the points in the red curve ranging from 6.5 to 7.2 (Fig. 2d, phase I) were bigger.

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Thank the reviewer for the comment.We have corrected "Fig.3C" to "Fig.3c" in the revised manuscript.
Thank the reviewer for the comment.We have corrected "Fig.3d" to "Fig.3c" in the revised manuscript.

7) G. Rampioni et al. (Chemical Communications 2018) also reported pioneering work in this
research line.This should be cited in the text.
Thank the reviewer for the comment.We have cited this paper as below.
[…] Supplementary Fig .21.(a) Species A′′ encapsulating fluorescein and pH-responsive molecules.(b) Yeast (species B).(c) Species C labelled with TR DHPE.Schematic diagram of A′′CB community with top view (d) and side view (e).(f) Merged image of laser scanning confocal images of A′′CB community taken with red and green channels, and white field.(g) Laser scanning confocal microscopy images of A′′CB community with green channel over time.Schematic diagram of CBA′′ community with top view (h) and side view (i).(j) Merged image of laser scanning confocal images of CBA′′ community taken with red and green channels, and white field.(k) Laser scanning confocal microscopy images of CBA′′ community with green channel over time.The scale bars were 20 μm in a, b and c, and 100 μm in f, g, j, and k.

Fig. R1.
Fig. R1.a, Time-dependent plot of sucrose concentrations released from species A (containing 400 mM sucrose) in the different environment solution pH.b, Plot of relative diffusion coefficient of sucrose at different environment solution pH, derived from (a).

Fig.
Fig. R4.a, The scheme of non-linear feedback network in the oscillation computational model II.b, Time-dependent plot of the negative number of the logarithm of ′C′ concentrations under different value of k1′ in oscillation computational model II.The value of k1′ was set as 1 and 10 respectively.
inside giant vesicles.By changing the surrounding pH, the artificial cells release sugar into the environment and the neighbor yeasts consume the sugar and release CO2 into the environment.As a consequence, the oscillation of pH change was generated in the society.The authors developed the society more complicated by increasing the species as 2 types of artificial cells and yeasts.Finally, the authors demonstrate the controlled communication between the species by giving spatial constraints.

Fig. R5 .
Fig. R5.The variation of yeast population density during oscillation process

Fig. 2 .
Fig. 2. Construction of two-species-community of pH-responsive artificial cells (containing sucrose) (speciesA) and yeast (species B) and its pH oscillatory environment.a, Schematic illustration of solution pH oscillation caused by the feedback between species A and B. b, A typical solution pH oscillation of two-species-community system as a function of time.Species A and B was 2.76×10 6 /mL and 4.36×10 6 /mL respectively.c, pH value of CO2 oversaturated (phase I, red curve) and saturated (phase II, blue curve) gadobutrol solution as a function of time.CO2 was injected into gadobutrol solution in phase I.The gadobutrol solution was saturated by CO2 from air in phase II.d, The initial sucrose concentration and sucrose concentration at the end of oscillation after 960 min.The sucrose concentrations were obtained by adding 10 % Triton-100 into the solution to release sucrose from species A. Data are presented as mean values ± SD, n = 3. e, Flow cytometry scatter plots of live-dead stained species B at longest oscillation time (960 min).Live yeasts (species B) were stained with FDA (green, in Q3), and dead yeasts were stained with PI (red, in Q1).Total number of particles counted was10,000.
". G. Rampioni et al. (Chemical Communications 2018) also reported pioneering work in this research line.This should be cited in the text.